BACKGROUND: The GenoType MTBDRsl v2 is a molecular test designed for the rapid detection of resistance to second-line anti-TB drugs in Mycobacterium tuberculosis complex (MTBC).OBJECTIVE: To assess the use of MTBDRsl in a programmatic setting and to describe the resistance patterns in a high HIV-TB-endemic area in South Africa.METHODS: We performed a retrospective data analysis of all MTBDRsl results in patients with newly diagnosed rifampicin-resistant TB (RR-TB). We compared its performance on direct testing of smear-positive and smear-negative specimens. Results were examined to observe the detected resistance-conferring mutations.RESULTS: Of 1873 RR-TB/multidrug-resistant TB (MDR-TB), 37.4% were smear-negative and 62.5% were smear-positive. Among smear-negative specimens, the MTBDRsl showed an inconclusive rate of 61.2%, while the inconclusive rate from smear-positive specimens was 6.6%. The most common mutation observed in case of fluoroquinolone resistance occurred at the gyrA gene, codon 90 (A90V) (61/158, 38.6%), and the most common mutation in injectable aminoglycoside resistance occurred in the rrs region, A1401G (71/108, 65.7%).CONCLUSION: In HIV-TB-prevalent settings, routine use of the MTBDRsl is more effective when performed directly on smear-positive specimens. In view of currently used injectable-free regimens, this test requires revision.